The present invention is a promoter sequence of 3-phosphogycerate kinase gene 2 of lactic acid-producing fungus Rhizopus oryzae and a method of expressing a gene of interest in fungal species.
The genus of Rhizopus is versatile in the production of biocatalysts such as glucoamylase and lipase and chemicals including L-(+)-lactic acid, fumaric acid, and ethanol. Rhizopus is the member of the order Mucorales, which is within the class Zygomycetes of the division Amastigomycota. Rhizopus oryzae (ATCC 9363) is the best lactic acid producer found in the Rhizopus genus, while Rhizopus delemar and Rhizopus niveus can produce significant amount of extracellular lipase. In addition, R. oryzae can also secrete large amount of glucoamylase in the solid culture for starch hydrolysis. Therefore, R. oryzae could be potentially a host for upgrading lactic acid production as well as foreign protein production. However, in the current literature, there is very limited information available on gene clones as well as gene regulatory elements (promoters) for R. oryzae. Less than nine gene clone and partial gene sequences are reported for R. oryzae, which include glucoamylase, ribosomal genes, and aspartic proteinase genes (GenBank Data Base).
The ability to genetically manipulate filamentous fungi largely depends on the successfulness to develop the transformation methods and gene expression systems. Transformation methods have been developed for filamentous fungi, in particular, Aspergillus nidulans and Neurospora crassa, including others such as Aspergillus niger, Aspergillus oryzae, Penicillium nalgiovense. To effectively direct the transcription or expression of an interested gene, strong gene regulating elements or promoters are required. These promoters can be isolated from the upstream sequences of strongly expressed gene clones. Phosphoglycerate kinase gene is one of the highly expressed genes found in yeast and filamentous fungi. This gene encodes some of the most abundant mRNA in the yeast cells, accounting for up to 5% of the total cellular protein expression. After the discovery and characterization of Saccharomyces cerevisiae gene, other phosphoglycerate kinase genes were also isolated from various fungal species such as Penicillium chrysogenum and Rhizopus niveus using S. cerevisiae phosphoglycerate kinase gene as homologous gene probe. However, only a few of phosphoglycerate kinase gene promoters were isolated and characterized, which were from S. cerevisiae, Trichoderma reesei, and R. niveus, among others.
To genetically manipulate R. oryzae, either for the purpose of metabolic pathway modification, conferring necessary traits such as acid tolerance and upgrading of lactic acid production, or producing biocatalyst of interest, high levels of mRNA expression are always desirable. Therefore, there is a need to isolate strong promoter sequences of R. oryzae and design/develop expression vectors, harboring the isolated phosphoglycerate kinase gene promoter.
The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 2 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.